1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
3. SUMMARY OF THE INVENTION
4. BRIEF DESCRIPTION OF THE DRAWINGS
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. THE OB-R VARIANT
5.2. EXPRESSION OF THE OB-R VARIANT
5.3. USES OF THE OB-R VARIANT POLYNUCLEOTIDE
5.3.1. DIAGNOSTIC USES OF OB-R VARIANT POLYNUCLEOTIDE
5.3.2. THERAPEUTIC USES OF THE OB-R VARIANT POLYNUCLEOTIDE
5.4. ACTIVATION OF TYROSINE KINASE PATHWAYS IN OBESITY
6. EXAMPLE: MOLECULAR CLONING OF AN OB-R VARIANT
7. DEPOSIT OF MICROORGANISMS
The present invention relates to a variant form of the receptor for the obese gene product. In particular, the invention relates to methods of detecting this receptor variant in cells and tissues of obese individuals. In addition, it relates to methods of inhibiting or down-regulating expression of this variant in cells to augment their responsiveness to weight regulation by leptin as well as methods of using compounds to directly activate signal transduction pathways associated with this ligand-receptor system.
Obesity is not only a nutritional disorder in Western societies, it is also a serious health concern because of its association with adult-onset diabetes, hypertension, and heart disease (Grundy, 1990, Disease-a-Month 36:645-696). While there was evidence to suggest that body weight was physiologically regulated, the molecular mechanism has remained elusive. However, animal studies have produced several mouse strains that contain single-gene mutations, resulting in an obese phenotype. One such recessive mutation is manifested in the ob/ob mice, and it is referred to as the obese (ob) mutation.
Zhang et al. (1994, Nature 372:425-432) describe the cloning and sequencing of the mouse ob gene and its human homolog. When an isolated gene fragment was used as a probe, it was shown to hybridize with RNA only in white adipose tissue by northern blot analysis, but no expression was detected in any other tissue. In addition, the coding sequence of the ob gene hybridized to all vertebrate genomic DNAs tested, indicating a high level of conservation of this molecule among vertebrates. The deduced amino acid sequences are 84% identical between human and mouse, and both molecules contain features of secreted proteins.
In an effort to understand the physiologic function of the ob gene, several independent research groups produced recombinant ob gene product in bacteria for in vivo testing (Pelleymounter et al., 1995, Science 269:540-543; Halaas et al., 1995, Science 269:543-546; Campfield et al., 1995, Science 269:546-549). When the Ob protein (also known as leptin) was injected into grossly obese mice, which possessed two mutant copies of the ob gene, the mice exhibited a reduced appetite and began to lose weight. In addition, these studies described a dual action of leptin in both reducing the animals"" food intake and in increasing their energy expenditure. Similarly, when normal mice received leptin, they also ate less than the untreated controls. More importantly, Campfield et al. (1995, Science 269:546-549) injected leptin directly into lateral ventricle, and observed a reduction in the animals"" food intake, suggesting that leptin acts on central neuronal networks to regulate feeding behavior and energy balance. Thus, this result provides evidence that the leptin receptor (also known as OB-R) is expressed by cells in the brain.
Recently, a leptin fusion protein was generated and used to screen for OB-R in a cDNA expression library prepared from mouse choroid plexus, a tissue that lines brain cavities termed ventricles (Tartalia, 1995, Cell 83:1263-1271). This approach led to the cloning of one form of the OB-R coding sequence, which reveals a single membrane-spanning receptor, sharing structural similarities with several Class I cytokine receptors, such as the gp130 signal-transducing component of the interleukin-6 receptor (Taga et al., 1989, Cell 58:573-581), the granulocyte-colony stimulating factor receptor (Fukunaga et al., 1990, Cell 61:341-350), and the leukemia inhibitory factor receptor (Gearing et al., 1991, EMBO J. 10:2839-2848). Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) demonstrate that OB-R mRNA is expressed in several tissues, including lung, kidney, total brain, choroid plexus and hypothalamus.
The reported mouse OB-R protein contains a relatively short intracellular cytoplasmic domain as compared with other Class I cytokine receptors. Subsequently, when cDNA encoding its human homolog was isolated from a human infant brain library, the predicted human protein sequence contains a much longer intracellular domain. In view of this finding, it was speculated that different forms of the receptor might exist (Barinaga, 1996, Science 271:29). However, prior to the present invention, there was no report on the identification of any variant forms of the OB-R in humans or how such molecules, if they exist, would relate to obesity.
Additionally, several studies have shown that ob gene expression is actually increased in obese humans (Considine et al., 1995, J. Clin. Invest. 95:2986-2988; Lonnquist et al., 1995, Nature Med. 1:950; Hamilton et al., 1995, Nature Med. 1:953). Moreover, the mutations in the mouse Ob gene were not detected in human mRNA. Therefore, taken collectively, these studies imply that decreased leptin levels are not the primary cause of obesity, and argue for the presence of a less responsive receptor in obese individuals. There remains a need to isolate such an OB-R variant for the design of therapeutics to augment weight regulation by leptin.
The present invention relates to a variant form of the human OB-R. In particular, it relates to the detection of this receptor variant in cells of obese individuals, and methods for treating obesity by targeting this variant.
The invention is based, in part, upon the Applicants"" discovery of human cDNA clones encoding a variant form of the OB-R. This receptor differs structurally from a reported OB-R with only three amino acid substitutions in the extracellular domain, but extensive diversity is observed in their intracellular cytoplasmic domains at the 3xe2x80x2 end. The cytoplasmic domain of the variant of the invention is both shorter and distinct in nucleotide sequence from the corresponding domain of the published form of OB-R. Therefore, a wide variety of uses are encompassed by the present invention, including but not limited to, the detection of the receptor variant in cells of obese individuals, methods to inhibit and/or down-regulate the expression of this receptor variant, gene therapy to replace the receptor variant in homozygous individuals, and direct activation of downstream signal transduction pathways in cells expressing the receptor variant for weight regulation.